DNA encoding type II IL-1 receptors

ABSTRACT

Type II IL-1 receptor (type II IL-1R) proteins, DNAs and expression vectors encoding type II IL-1R, and processes for producing type II IL-1R as products of recombinant cell culture, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/441,893, filedMay 16, 1995, which is a continuation of U.S. Ser. No. 08/242,211, filedMay 13, 1994, issued as U.S. Pat. No. 5,464,937, which is a divisionalof U.S. Ser. No. 08/091,519, filed Jul. 12, 1993, now U.S. Pat. No.5,350,683, which is a continuation of U.S. Ser. No. 07/701,415, filedMay 16, 1991, now abandoned, which is a continuation in part of U.S.Ser. No. 07/627,071, filed Dec. 13, 1990, now abandoned, which is acontinuation in part of U.S. Ser. No. 07/573,576, filed Aug. 24, 1990,now abandoned, which is a continuation in part of U.S. Ser. No.07/534,193, filed Jun. 5, 1990, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to cytokine receptors, and morespecifically, to Type II (B Cell) Interleukin-1 receptors.

Interleukin-1α (IL-1α) and Interleukin-1β and (IL-1β) are distantlyrelated polypeptide hormones which play a central role in the regulationof immune and inflammatory responses. These two proteins act on avariety of cell types and have multiple biological activities. Thediversity of biological activity ascribed to IL-1α and IL-1β is mediatedby specific plasma membrane receptors which bind both IL-1α and IL-1β.Due to the wide range of biological activities mediated by IL-1α andIL-1β it was originally believed that the IL-1 receptors should behighly conserved in a variety of species and expressed on a largevariety of cells.

Structural chracterization by ligand affinity cross-linking techniqueshas demonstrated that, despite their significant divergence in sequence,IL-1α and IL-1β bind to the same cell surface receptor molecule on Tcells and fibroblasts (Dower et al., Nature (London) 324:266, 1986; Birdet al., Nature (London) 324:263, 1986; Dower et al., Proc. Natl. Acad.Sci. USA 83:1060, 1986). The IL-1 receptor on murine and human T cellshas been identified by cDNA expression cloning and N-terminal sequenceanalysis as an integral membrane glycoprotein that binds IL-1α and IL-1βand has a molecular weight of 80,000 kDa (Sims et al., Science 241:585,1988; Sims et al., Proc. Natl. Acad. Sci. USA 86:8946, 1989).

It is now clear, however, that this 80 kDa IL-1 receptor protein doesnot mediate all the diverse biological effects of IL-1. Subsequentaffinity cross-linking studies indicate that IL-1 receptors on theEpstein Barr virus (EBV)-transformed human B cell lines VDS-O and 3B6,the EBV-positive Burkitt's lymphoma cell line Raji, and the murine pre-Bcell line 70Z/3, have a molecular weight of 60,000 to 68,000 kDa(Matsushima et al., J. Immunol. 136:4496, 1986; Bensimon et al., J.Immunol. 142:2290, 1989; Bensimon et al., J. Immunol. 143:1168, 1989;Horuk et al., J. Biol. Chem. 262:16275, 1987; Chizzonite et al., Proc.Natl. Acad. Sci. USA 86:8029, 1989; Bomsztyk et al., Proc. Natl. Acad.Sci. USA 86:8034, 1989). Moreover, comparison of the biochemicalproperties and kinetic analysis of the IL-1 receptor in the Raji B cellline with EL-4 murine T lymphoma cell line showed that Raji cells hadlower binding affinity but much higher receptor density per cell than asubclone of EL-4 T cells (Horuk et al., J. Biol. Chem. 262:16275, 1987).Raji cells also failed to internalize IL-1 and demonstrated alteredreceptor binding affinities with IL-1 analogs. (Horuk et al., J. Biol.Chem. 262:16275, 1987). These data suggest that the IL-1 receptorsexpressed on B cells (referred to herein as type II IL-1 receptors) aredifferent from IL-1 receptors detected on T cells and other cell types(referred to herein as type I IL-1 receptors).

In order to study the structural and biological characteristics of typeII IL-1R and the role played by type II IL-1R in the responses ofvarious cell populations to IL-1 stimulation, or to use type II IL-1Reffectively in therapy, diagnosis, or assay, homogeneous compositionsare needed. Such compositions are theoretically available viapurification of receptors expressed by cultured cells, or by cloning andexpression of genes encoding the receptors. Prior to the presentinvention, however, several obstacles prevented these goals from beingachieved.

First, no cell lines have previously been known to express high levelsof type II IL-1R constitutively and continuously, and cell lines knownto express type II IL-1R did so only in low numbers (500 to 2,000receptors/cell) which impeded efforts to purify receptors in amountssufficient for obtaining amino acid sequence information or generatingmonoclonal antibodies. The low numbers of receptors has also precludedany practical translation assay-based method of cloning.

Second, the significant differences in DNA sequence between type I IL-1Rand type II IL-1R has precluded cross-hybridization using a murine typeIL-1R cDNA (Bomsztyk et al., Proc. Natl. Acad. Sci. USA 86:8034, 1989,and Chizzonite et al., Proc. Natl. Acad. Sci. USA 86:8029, 1989).

Third, even if a protein composition of sufficient purity could beobtained to permit N-terminal protein sequencing, the degeneracy of thegenetic code may not permit one to define a suitable probe withoutconsiderable additional experimentation. Many iterative attempts may berequired to define a probe having the requisite specificity to identifya hybridizing sequence in a cDNA library. Although direct expressioncloning techniques avoid the need for repetitive screening usingdifferent probes of unknown specificity and have been useful in cloningother receptors (e.g., type I IL-1R), they are not sufficientlysensitive to be suitable for using in identifying type II IL-1R clonesfrom cDNA libraries derived from cells expressing low numbers of type IIIL-1R.

Thus, efforts to purify the type II IL-1R or to clone or express genesencoding type II IL-1R have been significantly impeded by lack ofpurified receptor, a suitable source of receptor mRNA, and by asufficiently sensitive cloning technique.

SUMMARY OF THE INVENTION

The present invention provides isolated type II IL-1R and isolated DNAsequences encoding type II IL-1R, in particular, human type II IL-1R, oranalogs thereof. Preferably, such DNA sequences are selected from thegroup consisting of (a) cDNA clones having a nucleotide sequence derivedfrom the coding region of a native type II IL-1R gene, such as clone 75;(b) DNA sequences capable of hybridization to the cDNA clones of (a)under moderately stringent conditions and which encode biologicallyactive IL-1R molecules; and (c) DNA sequences which are degenerate as aresult of the genetic code to the DNA sequences defined in (a) and (b)and which encode biologically active IL-1R molecules. The presentinvention also provides recombinant expression vectors comprising theDNA sequences defined above, recombinant type II IL-1R moleculesproduced using the recombinant expression vectors, and processes forproducing the recombinant type II IL-1R molecules utilizing theexpression vectors.

The present invention also provides substantially homogeneous proteincompositions comprising type II IL-1R.

The present invention also provides compositions for use in therapy,diagnosis, assay of type II IL-1R, or in raising antibodies to type IIIL-1R, comprising effective quantities of soluble native or recombinantreceptor proteins prepared according to the foregoing processes.

These and other aspects of the present invention will become evidentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scehmatic diagram of the expression plasmid pDC406. cDNAmolecules inserted at the Sal site are transcribed and translated usingregulatory elements derived from HIV and adenovirus. pDC406 containsorigins of replication derived from SV40, Epstein-Barr virus and pBR322.

FIG. 2 is a schematic diagram of the human and murine type II IL-1receptors and the various human and murine clones used to determine thesequences. Thin lines represent untranslated regions, while the codingregion is depicted by a box. The sections encoding the signal peptideare filled in; the transmembrane regions are cross-hatched; and thecytoplasmic portions are stippled. Potential N-linked glycosylationsites are marked by inverted triangles. The predictedimmunoglobulin-like disulfide bonds are also indicated by dashesconnecting two sulfide molecules (S—————S).

FIG. 3 compares the amino acid sequences of the human and murine type IIIL-1 receptors (as deduced from the cDNA clones) with the amino acidsequences of the human and murine type I IL-1 receptors (Sims et al.,Proc. Natl. Acad. Sci. USA 86:8946, 1989; Sims et al., Science 241:585,1988) and the amino acid sequences of the ST2 cellular gene (Tominaga,FEBS Lett. 258:301, 1989) and the B15R open reading frame of vacciniavirus (Smith and Chan, J. Gen. Virology 72:511, 1991). Numbering beginswith the initiating methionine. The predicted position of the signalpeptide cleavage in each sequences was determined according to themethod described by von Heijne, Nucl. Acids. Res. 14:4683, 1986, and isindicated by a gap between the putative signal peptide and the main bodyof the protein. The predicated transmembrane and cytoplasmic regions forthe type II IL-1 receptors are shown on the bottom line, and areseparated from one another by a gap. Residues conserved in all four IL-1receptor sequences are presented in white on a black background.Residues conserved in type II receptors that are also found in one ofthe other sequences are shaded; residues conserved in type I IL-1receptors that are found in one of the other sequences are boxed.Cysteine residues involved in forming the disulfide bonds characteristicof the immunolgobulin fold are marked with solid dots, while the extratwo pairs of cysteines found in the type I IL-1 receptor and in some ofthe other sequences are indicated by stars. The approximate boundariesof domains 1, 2 and 3 are indicated above the lines. The predictedsignal peptide cleavage in the type II IL-1 receptors follow Ala13,resulting in an unusually short signal peptide and an N-terminalextension of 12 (human) or 23 (mouse) amino acids beyond the pointcorresponding to the mature N-terminus of the human or mouse type I IL-1receptor. Other less favored but still acceptable sites of cleavage inthe murine type II IL-1 receptor are after Thr15 or Pro17. This sequencealignment was made by hand and does not represent an objectivelyoptimized alignment of the sequences. The nucleotide and amino acidsequences of the full length and soluble human and murine type II IL-1receptor cDNAs are also set forth in the Sequence Listing herein.

FIG. 4 shows an autoradiograph of an SDS/PAGE gel with crosslinked IL-1receptors. Cells expressing IL-1 receptors were cross-linked to¹²⁵I-IL-1 in the absence or presence of the cognate unlabeled IL-1competitor, extracted, electrophoresed and autoradiographed as describedin Example 6. Recombinant receptors were expressed transiently inCV1EBNA cells. The cell lines used for cross-linking to naturalreceptors were KB (ATCC CCL 1717) (for human type I IL-1R), CB23 (forhuman type II IL-1R), EL4 (ATCC TIB 39) (for murine type I IL-1R), and70Z/3 (ATCC TIB 158) (for murine type II IL-1R).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“IL-1” refers collectively to IL-1α and IL-1β.

“Type II Interleukin-1 receptor” and “type II IL-1R” refer to proteinswhich are capable of binding Interleukin-1 (IL-1) molecules and, intheir native configuration as mammalian plasma membrane proteins, play arole in transducing the signal provided by IL-1 to a cell. The maturefull-length human type II IL-1R is a glycoprotein having an apparentmolecular weight of approximately 60-68 kDa. Specific examples of typeII IL-1R proteins are shown in SEQ ID NO:1 and SEQ ID NO:12. As usedherein, the above terms include analogs or subunits of native type IIIL-1R proteins with IL-1-binding or signal transducing activity.Specifically included are truncated or soluble forms of type II IL-1Rprotein, as defined below. In the absence of any species designation,type II IL-1R refers generically to mammalian type II IL-1R, whichincludes, but is not limited to, human, murine, and bovine type IIIL-1R. Similarly, in the absence of any specific designation fordeletion mutants, the term type II IL-1R means all forms of type IIIL-1R, including mutants and analogs which possess type II IL-1Rbiological activity. “Interleukin-1 Receptor” or “IL-1R” referscollectively to type I IL-1 receptor and type II IL-1 receptor.

“Soluble type II IL-1R” as used in the context of the present inventionrefer to proteins, or substantially equivalent analogs, which aresubstantially similar to all or part of the extracellular region of anative type II IL-1R, and are secreted by the cell but retain theability to bind IL-1 or inhibit IL-1 signal transduction activity viacell surface bound IL-1R proteins. Soluble type II IL-1R proteins mayalso include part of the transmembrane region, provided that the solubletype II IL-1R protein is capable of being secreted from the cell.Specific examples of soluble type II IL-1R proteins include proteinshaving the sequence of amino acids 1-330 or amino acids 1-333 of SEQ IDNO:1 and amino acids 1-342 and amino acids 1-345 of SEQ ID NO:12.Inhibition of IL-1 signal transduction activity can be determined usingprimary cells or cells lines which express an endogenous IL-1R and whichare biologically responsive to IL-1 or which, when transfected withrecombinant IL-1R DNAs, are biologically responsive to IL-1. The cellsare then contacted with IL-1 and the resulting metabolic effectsexamined. If an effect results which is attributable to the action ofthe ligand, then the recombinant receptor has signal transductionactivity. Exemplary procedures for determining whether a polypeptide hassignal transduction activity are disclosed by Idzerda et al., J. Exp.Med. 171:861 (1990); Curtis et al., Proc. Natl. Acad. Sci. USA 86:3045(1989); Prywes et al., EMBO J. 5:2179 (1986) and Chou et al., J. Biol.Chem. 262:1842 (1987).

The term “isolated” or “purified”, as used in the context of thisspecification to define the purity of type II IL-1R protein or proteincompositions, means that the protein or protein composition issubstantially free of other proteins of natural or endogenous origin andcontains less than about 1% by mass of protein contaminants residual ofproduction processes. Such compositions, however, can contain otherproteins added as stabilizers, carriers, excipients or co-therapeutics.Type II IL-1R is “isolated” if it is detectable as a single protein bandin a polyacrylamide gel by silver staining.

The term “substantially similar,” when used to define either amino acidor nucleic acid sequences, means that a particular subject sequence, forexample, a mutant sequence, varies from a reference sequence by one ormore substitutions, deletions, or additions, the net effect of which isto retain biological activity of the type II IL-1R protein as may bedetermined, for example, in a type II IL-1R binding assays, such as isdescribed in Example 5 below. Alternatively, nucleic acid subunits andanalogs are “substantially similar” to the specific DNA sequencesdisclosed herein if: (a) the DNA sequence is derived from the codingregion of SEQ ID NO:1 or SEQ ID NO:12; (b) the DNA sequence is capableof hybridization to DNA sequences of (a) under moderately stringentconditions (25% formamide, 42° C., 2×SSC) or alternatively under morestringent conditions (50% formamide, 50° C., 2×SSC or 50% formamide, 42°C., 2×SSC) and which encode biologically active IL-1R molecules; or DNAsequences which are degenerate as a result of the genetic code to theDNA sequences defined in (a) or (b) and which encode biologically activeIL-1R molecules.

“Recombinant,” as used herein, means that a protein is derived fromrecombinant (e.g., microbial or mammalian) expression systems.“Microbial” refers to recombinant proteins made in bacterial or fungal(e.g., yeast) expression systems. As a product, “recombinant microbial”defines a protein essentially free of native endogenous substances andunaccompanied by associated native glycosylation. Protein expressed inmost bacterial cultures, e.g., E. coli, will be free of glycan; proteinexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells.

“Biologically active,” as used throughout the specification as acharacteristic of type II IL-1R, means either that a particular moleculeshares sufficient amino acid sequence similarity with SEQ ID NO:2 or SEQID NO:13 to be capable of binding detectable quantities of IL-1,preferably at least 0.01 nmoles IL-1 per nanomole type II IL-1R, or, inthe alternative, shares sufficient amino acid sequence similarity to becapable of transmitting an IL-1 stimulus to a cell, for example, as acomponent of a hybrid receptor construct. More preferably, biologicallyactive type II IL-1R within the scope of the present invention iscapable of binding greater than 0.1 nanomoles IL-1 per nanomolereceptor, and most preferably, greater than 0.5 nanomoles IL-1 pernanomole receptor.

“DNA sequence” refers to a DNA polymer, in the form of a separatefragment or as a component of a larger DNA construct, which has beenderived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. However, it will be evident that genomicDNA containing the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5′ or 3′ from the open reading frame,where the same do not interfere with manipulation or expression of thecoding regions.

“Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides.DNA sequences encoding the proteins provided by this invention areassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

“Recombinant expression vector” refers to a plasmid comprising atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

“Recombinant microbial expression system” means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E. coli or yeast such as S. cerevisiae, which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Isolation of cDNAs Encoding Type II IL-1R

In order to secure a human coding sequence, a DNA sequence encodinghuman type II IL-1R (see SEQ ID NO:1) was isolated from a cDNA libraryprepared using standard methods by reverse transcription ofpolyadenylated RNA isolated from the human B cell lymphoblastoid lineCB23, described by Benjamin & Dower, Blood 75:2017, 1990. Briefly, theCB23 cell line is an EBV-transformed cord blood (CB) lymphocyte cellline, which was derived using the methods described by Benjamin et al.,Proc. Natl. Acad. Sci. USA 81:3547, 1984.

The CB23 library was screened by modified direct expression of pooledcDNA fragments in the monkey kidney cell line CV-1/EBNA-1 using amammalian expression vector (pDC406) that includes regulatory sequencesderived from SV40 and human immunodeficiency virus (HIV), andEpstein-Barr virus (EBV). The CV-1/EBNA-1 cell line was derived bytransfection of the CV-1 cell line with the gene encoding Epstein-Barrvirus nuclear antigen-1 (EBNA-1) and constitutively expresses EBNA-1driven from the human CMV immediate-early enhancer/promoter. The EBNA-1gene allows the episomal replication of expression vectors such aspDC406 that contain the EBV origin of replication.

Transfectants expressing biologically active type II IL-1R wereinitially identified using a modified slide autoradiogaphic technique,substantially as described by Gearing et al., EMBO J. 8:3667, 1989.Briefly, CV-1/EBNA-1 cells were transfected with miniprep DNA in pDC406from pools of cDNA clones directly on glass slides and cultured for 2-3days to permit transient expression of type II IL-1R. The slidescontaining the transfected cells were then incubated with mediumcontaining ¹²⁵I-IL-1, washed to remove unbound labeled IL-1β, fixed withglutaraldehyde, and dipped in liquid photographic emulsion and exposedin the dark. After developing the slides, they were individuallyexamined with a microscope and positive cells expressing type II IL-1Rwere identified by the presence of autoradiographic silver grainsagainst a light background.

Using this approach, approximately 250,000 cDNAs were screened in poolsof approximately 3,000 cDNAs using the slide autoradiographic methoduntil assay of one transfectant pool showed multiple cells clearlypositive for IL-1β binding. This pool was then partitioned into pools of500 and again screened by slide autoradiography and a positive pool wasidentified. This pool was further partitioned into pools of 75 andscreened by plate binding assays analyzed by quantitation of bound¹²⁵I-IL-1β. The cells were scraped off and counted to determine whichpool of 75 was positive. Individual colonies from this pool of 75 werescreened until a single clone (clone 75) was identified which directedsynthesis of a surface protein with detectable IL-1β binding activity.This clone was isolated, and its insert was sequenced to determine thesequence of the human type II IL-1R cDNA clone 75 (SEQ ID NO:1). ThepDC406 cloning vector containing the human type II IL-1R cDNA,designated pHuTYPE II IL-1R 75, was deposited with the American TypeCulture Collection, Rockville, Md., USA (ATCC) on Jun. 5, 1990 underaccession number CRL 10478. The deposit was made under the conditions ofthe Budapest Treaty.

A probe may be constructed from the human sequence and used to screenvarious other mammalian cDNA libraries. cDNA clones which hybridized tothe human probe are then isolated and sequenced.

Like most mammalian genes, mammalian type II IL-1R is presumably encodedby multi-exon genes. Alternative mRNA constructs which can be attributedto different mRNA splicing events following transcription, and whichshare large regions of identity or similarity with the cDNAs claimedherein, are considered to be within the scope of the present invention.

Proteins and Analogs

The present invention provides isolated recombinant mammalian type IIIL-1R polypeptides. Isolated type II IL-1R polypeptides of thisinvention are substantially free of other contaminating materials ofnatural or endogenous origin and contain less than about 1% by mass ofprotein contaminants residual of production processes. The native humantype II IL-1R molecules are recovered from cell lysates as glycoproteinshaving an apparent molecular weight by SDS-PAGE of about 60-68kilodaltons (kDa). The type II IL-1R polypeptides of this invention areoptionally without associated native-pattern glycosylation.

Mammalian type 11 IL-1R of the present invention includes, by way ofexample, primate, human, murine, canine, feline, bovine, ovine, equine,caprine and porcine type II IL-1R. Mammalian type II IL-1R can beobtained by cross species hybridization, using a single stranded cDNAderived from the human type II IL-1R DNA sequence, for example, clone75, as a hybridization probe to isolate type II IL-1R cDNAs frommammalian cDNA libraries. DNA sequences which encode IL-IR-II, possiblyin the form of alternate splicing arrangements, can be isolated from thefollowing cells and tissues: B lymphoblastoid lines (such as CB23, CB33,Raji, RPMI1788, ARH77), resting and especially activated peripheralblood T cells, monocytes, the monocytic cell line THP1, neutrophils,bone marrow, placenta, endothelial cells, keratinocytes (especiallyactivated), and HepG2 cells.

Derivatives of type II IL-1R within the scope of the invention alsoinclude various structural forms of the primary protein which retainbiological activity. Due to the presence of ionizable amino and carboxylgroups, for example, a type II IL-1R protein may be in the form ofacidic or basic salts, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Covalent derivatives are prepared bylinking particular functional groups to type II IL-1R amino acid sidechains or at the N- or C-termini. Other derivatives of type II IL-1Rwithin the scope of this invention include covalent or aggregativeconjugates of type II IL-1R or its fragments with other proteins orpolypeptides, such as by synthesis in recombinant culture as N-terminalor C-terminal fusions. For example, the conjugated peptide may be a asignal (or leader) polypeptide sequence at the N-terminal region of theprotein which co-translationally or post-translationally directstransfer of the protein from its site of synthesis to its site offunction inside or outside of the cell membrane or wall (e.g., the yeastα-factor leader). Type II IL-1R protein fusions can comprise peptidesadded to facilitate purification or identification of type II IL-1R(e.g., poly-His). The amino acid sequence of type II IL-1R can also belinked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hoppet al., Bio/Technology 6:1204,1988.) The latter sequence is highlyantigenic and provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant protein. This sequence is also specificallycleaved by bovine mucosal enterokinase at the residue immediatelyfollowing the Asp-Lys pairing. Fusion proteins capped with this peptidemay also be resistant to intracellular degradation in E. coli.

Type II IL-1R derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of IL-1 or other binding ligands. type II IL-1Rderivatives may also be obtained by cross-linking agents, such asM-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. Type II IL-1R proteins may also becovalently bound through reactive side groups to various insolublesubstrates, such as cyanogen bromide-activated, bisoxirane-activated,carbonyldiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, type II IL-1R may be used toselectively bind (for purposes of assay or purification) anti-type IIIL-1R antibodies or IL-1.

The present invention also includes type I IL-1R with or withoutassociated native-pattern glycosylation. Type II IL-1R expressed inyeast or mammalian expression systems, e.g., COS-7 cells, may be similaror slightly different in molecular weight and glycosylation pattern thanthe native molecules, depending upon the expression system. Expressionof type II IL-1R DNAs in bacteria such as E. coli providesnon-glycosylated molecules. Functional mutant analogs of mammalian typeII IL-1R having inactivated N-glycosylation sites can be produced byoligonucleotide synthesis and ligation or by site-specific mutagenesistechniques. These analog proteins can be produced in a homogeneous,reduced-carbohydrate form in good yield using yeast expression systems.N-glycosylation sites in eukaryotic proteins are characterized by theamino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro, andZ is Ser or Thr. In this sequence, asparagine provides a side chainamino group for covalent attachment of carbohydrate. Examples ofN-glycosylation sites in human type II IL-1R are amino acids 66-68,72-74, 112-114, 219-221, and 277-279 in SEQ ID NO:1. Such sites can beeliminated by substituting another amino acid for Asn or for residue Z,deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, oran amino acid other than Asn between Asn and A₁.

Type II IL-1R derivatives may also be obtained by mutations of type lIIL-1R or its subunits. A type II IL-1R mutant, as referred to herein, isa polypeptide homologous to type II IL-1R but which has an amino acidsequence different from native type II IL-1R because of a deletion,insertion or substitution.

Bioequivalent analogs of type II IL-1R proteins may be constructed by,for example, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues can be deleted orreplaced with other amino acids to prevent formation of unnecessary orincorrect intramolecular disulfide bridges upon renaturation. Otherapproaches to mutagenesis involve modification of adjacent dibasic aminoacid residues to enhance expression in yeast systems in which KEX2protease activity is present. Generally, substitutions should be madeconservatively; i.e., the most preferred substitute amino acids arethose having physiochemical characteristics resembling those of theresidue to be replaced. Similarly, when a deletion or insertion strategyis adopted, the potential effect of the deletion or insertion onbiological activity should be considered. Substantially similarpolypeptide sequences, as defined above, generally comprise a likenumber of amino acids sequences, although C-terminal truncations for thepurpose of constructing soluble type II IL-1Rs will contain fewer aminoacid sequences. In order to preserve the biological activity of type IIIL-1Rs, deletions and substitutions will preferably result in homologousor conservatively substituted sequences, meaning that a given residue isreplaced by a biologically similar residue. Examples of conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another, or substitutions of onepolar residue for another, such as between Lys and Arg; Glu and Asp; orGln and Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Moreover, particular amino aciddifferences between human, murine and other mammalian type II IL-1Rs issuggestive of additional conservative substitutions that may be madewithout altering the essential biological characteristics of type IIIL-1R.

Subunits of type II IL-1R may be constructed by deleting terminal orinternal residues or sequences. The present invention contemplates, forexample, C terminal deletions which result in soluble type II IL-1Rconstructs corresponding to all or part of the extracellular region oftype II IL-1R. The resulting protein preferably retains its ability tobind IL-1. Particularly preferred sequences include those in which thetransmembrane region and intracellular domain of type II IL-1R aredeleted or substituted with hydrophilic residues to facilitate secretionof the receptor into the cell culture medium. Soluble type II IL-1Rproteins may also include part of the transmembrane region, providedthat the soluble type II IL-1R protein is capable of being secreted fromthe cell. For example, soluble human type II IL-1R may comprise thesequence of amino acids 1-333 or amino acids 1-330 of SEQ ID NO:1 andamino acids 1-345 and amino acids 1-342 of SEQ ID NO:12. Alternatively,soluble type II IL-1R proteins may be derived by deleting the C-terminalregion of a type II IL-1R within the extracellular region which are notnecessary for IL-1 binding. For example, C-terminal deletions may bemade to proteins having the sequence of SEQ ID NO:1 and SEQ ID NO:12following amino acids 313 and 325, respectively. These amino acids arecysteines which are believed to be necessary to maintain the tertiarystructure of the type II IL-1R molecule and permit binding of the typeII IL-1R molecule to IL-1. Soluble type II IL-1R constructs areconstructed by deleting the 3′-terminal region of a DNA encoding thetype II IL-1R and then inserting and expressing the DNA in appropriateexpression vectors. Exemplary methods of constructing such solubleproteins are described in Examples 2 and 4. The resulting soluble type IIL-1R proteins are then assayed for the ability to bind IL-1, asdescribed in Example 5. Both the DNA sequences encoding such solubletype II IL-1Rs and the biologically active soluble type II IL-1Rproteins resulting from such constructions are contemplated to be withinthe scope of the present invention.

Mutations in nucleotide sequences constructed for expression of analogtype II IL-1R must, of course, preserve the reading frame phase of thecoding sequences and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structures such as loopsor hairpins which would adversely affect translation of the receptormRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed type II IL-1R mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes type II IL-1Rwill be expressed in the final product, for example, nucleotidesubstitutions may be made to enhance expression, primarily to avoidsecondary structure loops in the transcribed mRNA (see EPA 75,444A,incorporated herein by reference), or to provide codons that are morereadily translated by the selected host, e.g., the well-known E. colipreference codons for E. coli expression.

Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene15 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith etal. (Genetic Engineering: Principles and Methods, Plenum Press, 1981);and U.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques,and are incorporated by reference herein.

Both monovalent forms and polyvalent forms of type II IL-1R are usefulin the compositions and methods of this invention. Polyvalent formspossess multiple type II IL-1R binding sites for IL-1 ligand. Forexample, a bivalent soluble type II IL-1R may consist of two tandemrepeats of the extracellular region of type II IL-1R, separated by alinker region. Alternate polyvalent forms may also be constructed, forexample, by chemically coupling type II IL-1R to any clinicallyacceptable carrier molecule, a polymer selected from the groupconsisting of Ficoll, polyethylene glycol or dextran using conventionalcoupling techniques. Alternatively, type II IL-1R may be chemicallycoupled to biotin, and the biotin-type II IL-1R conjugate then allowedto bind to avidin, resulting in tetravalent avidin/biotin/type II IL-1Rmolecules. Type II IL-1R may also be covalently coupled to dinitrophenol(DNP) or trinitrophenol (TNP) and the resulting conjugate precipitatedwith anti-DNP or anti-TNP-IgM, to form decameric conjugates with avalency of 10 for type II IL-1R binding sites.

A recombinant chimeric antibody molecule may also be produced havingtype II IL-1R sequences substituted for the variable domains of eitheror both of the immunoglubulin molecule heavy and light chains and havingunmodified constant region domains. For example, chimeric type IIIL-1R/IgG₁ may be produced from two chimeric genes—a type II IL-1R/humanκ light chain chimera (type II IL-1R/C₇₈ ) and a type II IL-1R/human γ1heavy chain chimera (type II IL-1R/C₆₅ ₋₁). Following transcription andtranslation of the two chimeric genes, the gene products assemble into asingle chimeric antibody molecule having type II IL-1R displayedbivalently. Such polyvalent forms of type II IL-1R may have enhancedbinding affinity for IL-1 ligand. Additional details relating to theconstruction of such chimeric antibody molecules are disclosed in WO89/09622 and EP 315062.

Expression of Recombinant Type II IL-1R

The present invention provides recombinant expression vectors to amplifyor express DNA encoding type II IL-1R. Recombinant expression vectorsare replicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding mammalian type II IL-1R or bioequivalent analogsoperably linked to suitable transcriptional or translational regulatoryelements derived from mammalian, microbial, viral or insect genes. Atranscriptional unit generally comprises an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements may include an operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants mayadditionally be incorporated. DNA regions are operably linked when theyare functionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operably linked to DNA for a polypeptideif it is expressed as a precursor which participates in the secretion ofthe polypeptide; a promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto permit translation. Generally, operably linked means contiguous and,in the case of secretory leaders, contiguous and in reading frame.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

DNA sequences encoding mammalian type II IL-1Rs which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA; however,premature termination of transcription may be desirable, for example,where it would result in mutants having advantageous C-terminaltruncations, for example, deletion of a transmembrane region to yield asoluble receptor not bound to the cell membrane. Due to code degeneracy,there can be considerable variation in nucleotide sequences encoding thesame amino acid sequence. Other embodiments include sequences capable ofhybridizing to clone 75 under moderately stringent conditions (50° C.,2×SSC) and other sequences hybridizing or degenerate to those whichencode biologically active type II IL-1R polypeptides.

Recombinant type II IL-1R DNA is expressed or amplified in a recombinantexpression system comprising a substantially homogeneous monoculture ofsuitable host microorganisms, for example, bacteria such as E. coli oryeast such as S. cerevisiae, which have stably integrated (bytransformation or transfection) a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unit as acomponent of a resident plasmid. Generally, cells constituting thesystem are the progeny of a single ancestral transformant. Recombinantexpression systems as defined herein will express heterologous proteinupon induction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

Transformed host cells are cells which have been transformed ortransfected with type II IL-1R vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express type II IL-1R, buthost cells transformed for purposes of cloning or amplifying type IIIL-1R DNA do not need to express type II IL-1R. Expressed type II IL-1Rwill be deposited in the cell membrane or secreted into the culturesupernatant, depending on the type II IL-1R DNA selected. Suitable hostcells for expression of mammalian type II IL-1R include prokaryotes,yeast or higher eukaryotic cells under the control of appropriatepromoters. Prokaryotes include gram negative or gram positive organisms,for example E. coli or bacilli. Higher eukaryotic cells includeestablished cell lines of mammalian origin as described below. Cell-freetranslation systems could also be employed to produce mammalian type IIIL-1R using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described byPouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NewYork, 1985), the relevant disclosure of which is hereby incorporated byreference.

Prokaryotic expression hosts may be used for expression of type II IL-1Rthat do not require extensive proteolytic and disulfide processing.Prokaryotic expression vectors generally comprise one or more phenotypicselectable markers, for example a gene encoding proteins conferringantibiotic resistance or supplying an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimurium, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphyolococcus,although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Recombinant type II IL-1R proteins may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding type II IL-1R, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 or URA3gene, which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, and a promoter derived from ahighly expressed yeast gene to induce transcription of a structuralsequence downstream. The presence of the TRP1 or URA3 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan oruracil.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pUC18for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil or URA+tranformants in medium consisting of 0.67% YNB, with amino acids andbases as described by Sherman et al., Laboratory Course Manual forMethods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1986.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% or 4% glucose supplemented with 80 μg/ml adenine and 80μg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustionof medium glucose. Crude yeast supernatants are harvested by filtrationand held at 4° C. prior to further purification.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells is particularly preferred because suchproteins are generally correctly folded, appropriately modified andcompletely functional. Examples of suitable mammalian host &ell linesinclude the COS-7 lines of monkey kidney cells, described by Gluzman(Cell 23:175, 1981), and other cell lines capable of expressing anappropriate vector including, for example, L cells, C127, 3T3, Chinesehamster ovary (CHO), HeLa and BHK cell lines. Mammalian expressionvectors may comprise nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, and other 5′ or 3′ flanking nontranscribed sequences, and 5′or 3′ nontranslated sequences, such as necessary ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988).

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind 3 site toward the Bgl1 site located inthe viral origin of replication is included. Further, mammalian genomictype II IL-1R promoter, control and/or signal sequences may be utilized,provided such control sequences are compatible with the host cellchosen. Additional details regarding the use of a mammalian highexpression vector to produce a recombinant mammalian type II IL-1R areprovided in Examples 2 below. Exemplary vectors can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in, C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986).

In preferred aspects of the present invention, recombinant expressionvectors comprising type II IL-1R cDNAs are stably integrated into a hostcell's DNA. Elevated levels of expression product is achieved byselecting for cell lines having amplified numbers of vector DNA. Celllines having amplified numbers of vector DNA are selected, for example,by transforming a host cell with a vector comprising a DNA sequencewhich encodes an enzyme which is inhibited by a known drug. The vectormay also comprise a DNA sequence which encodes a desired protein.Alternatively, the host cell may be co-transformed with a second vectorwhich comprises the DNA sequence which encodes the desired protein. Thetransformed or co-transformed host cells are then cultured in increasingconcentrations of the known drug, thereby selecting for drug-resistantcells. Such drug-resistant cells survive in increased concentrations ofthe toxic drug by over-production of the enzyme which is inhibited bythe drug, frequently as a result of amplification of the gene encodingthe enzyme. Where drug resistance is caused by an increase in the copynumber of the vector DNA encoding the inhibitable enzyme, there is aconcomitant co-amplification of the vector DNA encoding the desiredprotein (e.g., type II IL-1R) in the host cell's DNA.

A preferred system for such co-amplification uses the gene fordihydrofolate reductase (DHFR), which can be inhibited by the drugmethotrexate (MTX). To achieve co-amplification, a host cell which lacksan active gene encoding DHFR is either transformed with a vector whichcomprises DNA sequence encoding DHFR and a desired protein, or isco-transformed with a vector comprising a DNA sequence encoding DHFR anda vector comprising a DNA sequence encoding the desired protein. Thetransformed or co-transformed host cells are cultured in mediacontaining increasing levels of MTX, and those cells lines which surviveare selected.

A particularly preferred co-amplification system uses the gene forglutamine synthetase (GS), which is responsible for the synthesis ofglutamine from glutamate and ammonia using the hydrolysis of ATP to ADPand phosphate to drive the reaction. GS is subject to inhibition by avariety of inhibitors, for example methionine sulphoximine (MSX). Thus,type II IL-1R can be expressed in high concentrations by co-amplifyingcells transformed with a vector comprising the DNA sequence for GS and adesired protein, or co-transformed with a vector comprising a DNAsequence encoding GS and a vector comprising a DNA sequence encoding thedesired protein, culturing the host cells in media containing increasinglevels of MSX and selecting for surviving cells. The GS co-amplificationsystem, appropriate recombinant expression vectors and cells lines, aredescribed in the following PCT applications: WO 87/04462, WO 89/01036,WO 89/10404 and WO 86/05807.

Recombinant proteins are preferably expressed by co-amplification ofDHFR or GS in a mammalian host cell, such as Chinese Hamster Ovary (CHO)cells, or alternatively in a murine myeloma cell line, such asSP2/0-Ag14 or NSO or a rat myeloma cell line, such as YB2/3.0-Ag20,disclosed in PCT applications WO/89/10404 and WO 86/05807.

A preferred eukaryotic vector for expression of type II IL-1R DNA isdisclosed below in Example 2. This vector, referred to as pDC406, wasderived from the mammalian high expression vector pDC201 and containsregulatory sequences from SV40, HIV and EBV.

Purification of Recombinant Type II IL-1 R

Purified mammalian type II IL-lRs or analogs are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, which are then purifiedfrom culture media or cell extracts.

For example, supernatants from systems which secrete recombinant solubletype II IL-1R protein into culture media can be first concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. For example, a suitable affinity matrix cancomprise an IL-1 or lectin or antibody molecule bound to a suitablesupport. Alternatively, an anion exchange resin can be employed, forexample, a matrix or substrate having pendant diethylaminoethyl (DEAE)groups. The matrices can be acrylamide, agarose, dextran, cellulose orother types commonly employed in protein purification. Alternatively, acation exchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a type II L-1R composition. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(RPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian type II IL-1R can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express soluble mammalian type II IL-1R as asecreted protein greatly simplifies purification. Secreted recombinantprotein resulting from a large-scale fermentation can be purified bymethods analogous to those disclosed by Urdal et al. (J. Chromatog.296:171, 1984). This reference describes two sequential, reversed-phaseHPLC steps for purification of recombinant human GM-CSF on a preparativeHPLC column.

Human type II IL-1R synthesized in recombinant culture is characterizedby the presence of non-human cell components, including proteins, inamounts and of a character which depend upon the purification stepstaken to recover human type II IL-1R from the culture. These componentsordinarily will be of yeast, prokaryotic or non-human higher eukaryoticorigin and preferably are present in innocuous contaminant quantities,on the order of less than about 1 percent by weight. Further,recombinant cell culture enables the production of type II IL-1R free ofproteins which may be normally associated with type II IL-1R as it isfound in nature in its species of origin, e.g. in cells, cell exudatesor body fluids.

Therapeutic Administration of Recombinant Soluble Type II IL-1R

The present invention provides methods of using therapeutic compositionscomprising an effective amount of soluble type II IL-1R proteins and asuitable diluent and carrier, and methods for suppressing IL-1-dependentimmune responses in humans comprising administering an effective amountof soluble type II IL-1R protein.

For therapeutic use, purified soluble type II IL-1R protein isadministered to a patient, preferably a human, for treatment in a mannerappropriate to the indication. Thus, for example, soluble type II IL-1Rprotein compositions can be administered by bolus injection, continuousinfusion, sustained release from implants, or other suitable technique.Typically, a soluble type II IL-1R therapeutic agent will beadministered in the form of a composition comprising purified protein inconjunction with physiologically acceptable carriers, excipients ordiluents. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the type II IL-1R with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with conspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents. Appropriate dosages can be determined in trials; generally,shuIL-1R dosages of from about 1 ng/kg/day to about 10 mg/kg/day, andmore preferably from about 500 μg/kg/day to about 5 mg/kg/day, areexpected to induce a biological effect.

Because IL-1R-I and type II IL-1R proteins both bind to IL-1, solubletype II IL-1R proteins are expected to have similar, if not identical,therapeutic activities. For example, soluble human type II IL-1R can beadministered, for example, for the purpose of suppressing immuneresponses in a human. A variety of diseases or conditions are caused byan immune response to alloantigen, including allograft rejection andgraft-versus-host reaction. In alloantigen-induced immune responses,shuIL-1R suppresses lymphoproliferation and inflammation which resultupon activation of T cells. shuIl-1R can therefore be used toeffectively suppress alloantigen-induced immune responses in theclinical treatment of, for example, rejection of allografts (such asskin, kidney, and heart transplants), and graft-versus-host reactions inpatients who have received bone marrow transplants.

Soluble human type II IL-1R can also be used in clinical treatment ofautoimmune dysfunctions, such as rheumatoid arthritis, diabetes andmultiple sclerosis, which are dependent upon the activation of T cellsagainst antigens not recognized as being indigenous to the host.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Isolation of cDNA Encoding Human Type II IL-1R byDirect Expression of Active Protein in CV-1/EBNA-1 Cells

A. Radiolabeling of rIL-1β. Recombinant human IL-1β was prepared byexpression in E. coli and purification to homogeneity as described byKronheim et al. (Bio/Technology 4:1078, 1986). The IL-1β was labeledwith di-iodo (¹²⁵I) Bolton-Hunter reagent (New England Nuclear,Glenolden, Pa.). Ten micrograms (0.57 nmol) of protein in 10 uL ofphosphate (0.015 mol/L)-buffered saline (PBS; 0.15 mol/L), pH 7.2, wasmixed with 10 uL of sodium borate (0.1 mol/L)-buffered saline (0.15mol/L), pH 8.5, and reacted with 1 mCi (0.23 nmol) of Bolton-Hunterreagent according to the manufacturer's instructions for 12 hours at 8°C. Subsequently, 30 uL of 2% gelatin and 5 uL of 1 mol/L glycine ethylester were added, and the protein was separated from unreactedBolton-Hunter reagent on a 1 mL bed volume Biogel™ P6 column (Bio-RadLaboratoreis, Richmond, Calif.). Routinely, 50% to 60% incorporation oflabel was observed. Radioiodination yielded specific activities in therange of 1×10¹⁵ to 5×10¹⁵ cpm/mmol-1 (0.4 to 2 atoms I per moleculeprotein), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) revealed a single labeled polypeptide of 17.5 kD, consistantwith previously reported values for IL-1. The labeled protein wasgreater than 98% TCA precipitable, indicating that the ¹²⁵I wascovalently bound to protein.

B. Construction and Screening of CB23 cDNA library. A CB23 library wasconstructed and screened by direct expression of pooled cDNA clones inthe monkey kidney cell line CV-1/EBNA-1 (which was derived bytransfection of the CV-1 cell line with the gene encoding EBNA-1, asdescribed below) using a mammalian expression vector (pDC406) thatincludes regulatory sequences from SV40, human immunodeficiency virus(HIV), and Epstein-Barr virus (EBV). The CV-1/EBNA-1 cell lineconstitutively expresses EBY nuclear antigen-1 driven from the humancytomegalovirus (CMV) immediate-early enhancer/promoter and thereforeallows the episomal replication of expression vectors such as pDC406that contain the EBV origin of replication. The expression vector usedwas pDC406, a derivative of HAV-EO, described by Dower et al., J.Immunol. 142:4314, 1989), which is in turn a derivative of pDC201 andallows high level expression in the CV-1/EBNA-1 cell line. pDC406differs from HAV-EO (Dower et al., supra) by the deletion of the intronpresent in the adenovirus 2 tripartite leader sequence in HAV-EO (seedescription of pDC303 below).

The CB23 cDNA library was constructed by reverse transcription ofpoly(A)⁺ mRNA isolated from total RNA extracted from the human B celllymphoblastoid line CB23 (Benjamin & Dower, Blood 75:2017, 1990)substantially as described by Ausubel et al., eds., Current Protocols inMolecular Biology, Vol. 1, 1987. The CB23 cell line is anEBV-transformed cord blood (CB) lymphocyte cell line, which was derivedby using the methods described by Benjamin et al., Proc. Natl. Acad.Sci. USA 81:3547, 1984. Poly(A)⁺ mRNA was isolated by oligo dT cellulosechromatography and double-stranded cDNA was made substantially asdescribed by Gubler and Hoffman, Gene 25:263, 1983. Briefly, thepoly(A)⁺ mRNA was converted to an RNA-cDNA hybrid with reversetranscriptase using random hexanucleotides as a primer. The RNA-cDNAhybrid was then converted into double-stranded cDNA using RNAase H incombination with DNA polymerase I. The resulting double stranded cDNAwas blunt-ended with T4 DNA polymerase. The following two unkinasedoligonucleotides were annealed and blunt end ligated with DNA ligase tothe ends of the resulting blunt-ended cDNA as described by Haymerle, etal., Nucleaic Acids Research, 14: 8615, 1986.

SEQ ID NO:3 5′-TCG ACT GGA ACG AGA CGA CCT GCT -3′ SEQ ID NO:43′-     GA CCT TGC TCT GCT GGA CGA -5′    <SalI>

In this case only the 24-mer oligo will ligate onto the cDNA. Thenon-ligated oligos were removed by gel filtration chromatography at 68°C., leaving 24 nucleotide non-self-complementary overhangs on the cDNA.The same procedure was used to convert the 5′ ends of SalI-cut mammalianexpression vector pDC406 to 24 nucleotide overhangs complementary tothose added to the cDNA. Optimal proportions of adaptored vector andcDNA were ligated in the presence of T4 polynucleotide kinase. Dialyzedligation mixtures were electroporated into E. coli strain DH5α.Approximately 3.9×10⁶ clones were generated and plated in pools ofapproximately 3,000. A sample of each pool was used to prepare frozenglycerol stocks and a sample was used to obtain a pool of plasmid DNA.

The pooled DNA was then used to transfect a sub-confluent layer ofmonkey CV-1/EBNA-1 cells using DEAE-dextran followed by chloroquinetreatment, similar to that described by Luthman et al., Nucl. Acids Res.11:1295 (1983) and McCutchan et al., J. Natl. Cancer Inst. 41:351(1986). CV-1/EBNA-1 cells were derived as follows. The CV-1/EBNA-1 cellline constitutively expresses EBV nuclear antigen-1 driven from the CMVimmediate-early enhancer/promoter. The African Green Monkey kidney cellline, CV-1 (ATCC CCL 70, was cotransfected with 5 μg of pSV2gpt(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981) and 25 μg ofpDC303/EBNA-1 using a calcium phosphate coprecipitation technique(Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley,New York, 1987). pDC303/EBNA-1 was constructed from pDC302 (Mosley etal., Cell 59:335, 1989) in two steps. First, the intron present in theadenovirus tripartite leader sequence was deleted by replacing a PvuIIto ScaI fragment spanning the intron with the following syntheticoligonucleotide pair to create plasmid pDC303:

SEQ ID NO:5 5′-CTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGT-3′

SEQ ID NO:6 3′-GACAACCCGAGCGCCAACTCCTGTTTGAGAAGCGCCAGAAAGGTCA-5′

Second, a HindIII-AhaII restriction fragment encoding Epstein-Barr virusnuclear antigen I (EBNA-1), and consisting essentially of EBVcoordinates 107,932 to 109,894 (Baer et al., Nature 310:207, 1984), wasthen inserted into the multiple cloning site of pDC303 to create theplasmid pDC303/EBNA-1. The transfected cells were grown in the presenceof hypoxanthine, aminopterin, thymidine, xanthine, and mycophenolic acidaccording to standard methods (Ausubel et al., supra; Mulligan & Berg,supra) to select for the cells that had stably incorporated thetransfected plasmids. The resulting drug resistant colonies wereisolated and expanded individually into cell lines for analysis. Thecell lines were screened for the expression of functional EBNA-1. Onecell line, clone 68, was found to express EBNA-1 using this assay, andwas designated CV-1/EBNA-1.

In order to transfect the CV-1/EBNA-1 cells with the cDNA library, thecells were maintained in complete medium (Dulbecco's modified Eagle'smedia (DMEM) containing 10% (v/v) fetal calf serum (FCS), 50 U/mlpenicillin, 50 U/ml streptomycin, 2 mM L-glutamine) and were plated at adensity of 2×10⁵ cells/well in either 6 well dishes (Falcon) or singlewell chambered slides (Lab-Tek). Both dishes and slides were pretreatedwith 1 ml human fibronectin (10 ug/ml in PBS) for 30 minutes followed by1 wash with PBS. Media was removed from the adherent cell layer andreplaced with 1.5 ml complete medium containing 66.6 μM chloroquinesulfate. 0.2 mls of DNA solution (2 μg DNA, 0.5 mg/ml DEAE-dextran incomplete medium containing chloroquine) was then added to the cells andincubated for 5 hours. Following the incubation, the media was removedand the cells shocked by addition of complete medium containing 10% DMSOfor 2.5 to 20 minutes followed by replacement of the solution with freshcomplete medium. The cells were grown in culture to permit transientexpression of the inserted sequences. These conditions led to an 80%transfection frequency in surviving CV-1/EBNA-1 cells.

After 48 to 72 hours, transfected monolayers of CV-1/EBNA cells wereassayed for expression of IL-1 binding proteins by bindingradioiodinated IL-1β prepared as described above by slideautoradiography. Transfected CV-1/EBNA-1 cells were washed once withbinding medium (RPMI medium 1640 containing 25 mg/ml bovine serumalbumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES, pH 7.2, and 50 mg/mlnonfat dry milk (NFDM)) and incubated for 2 hours at 4° C. with 1 mlbinding medium+NFDM containing 3×10⁻⁹ M ¹²⁵I-IL-1β. After incubation,cells in the chambered slides were washed three times with bindingbuffer+NFDM, followed by 2 washes with PBS, pH 7.3, to remove unbound¹²⁵I-IL-1β. The cells were fixed by incubating for 30 minutes at roomtemperature in 10% glutaraldehyde in PBS, pH 7.3, washed twice in PBS,and air dried. The slides were dipped in Kodak GTNB-2 photographicemulsion (6×dilution in water) and exposed in the dark for 48 hours to 7days at 4° C. in a light proof box. The slides were then developed forapproximately 5 minutes in Kodak D19 developer (40 g/500 ml water),rinsed in water and fixed in Agfa G433C fixer. The slides wereindividually examined with a microscope at 25-40×magnification andpositive cells expressing type II IL-1R were identified by the presenceof autoradiographic silver grains against a light background.

Cells in the 6 well plates were washed once with binding buffer+NFDMfollowed by 3 washings with PBS, pH 7.3, to remove unbound ¹²⁵I-IL-1β.The bound cells were then trypsinized to remove them from the plate andbound ¹²⁵I-IL-1β were counted on a beta counter.

Using the slide autoradiography approach, approximately 250,000 cDNAswere screened in pools of approximately 3,000 cDNAs until assay of onetransfectant pool showed multiple cells clearly positive for IL-1βbinding. This pool was then partitioned into pools of 500 and againscreened by slide autoradiography and a positive pool was identified.This pool was further partitioned into pools of 75, plated in 6-wellplates and screened by plate binding assays analyzed by quantitation ofbound ¹²⁵I-IL-1β. The cells were scraped off the plates and counted todetermine which pool of 75 was positive. Individual colonies from thispool of 75 were screened until a single clone (clone 75) was identifiedwhich directed synthesis of a surface protein with detectable IL-1binding activity. This clone was isolated, and its insert was sequencedto determine the sequence of the human type II IL-1R cDNA clone 75. ThepDC406 cloning vector containing the human type II IL-1R cDNA clone 75,designated pHuIL-1R-II 75, was deposited with the American Type CultureCollection, Rockville, Md., USA (ATCC) on Jun. 5, 1990 under accessionnumber CRL 10478. The Sequence Listing setting forth the nucleotide (SEQID No:1) and predicted amino acid sequences of clone 75 (SEQ ID No:1 andSEQ ID NO:2) and associated information appears at the end of thespecification immediately prior to the claims.

Example 2 Construction and Expression of cDNAs Encoding Human SolubleType II IL-1R

A cDNA encoding a soluble human type II IL-1R (having the sequence ofamino acids -13-333 of SEQ ID NO:1) was constructed by polymerase chainreaction (PCR) amplification using the full length type II IL-1R cDNAclone 75 (SEQ ID NO:1) in the vector pDC406 as a template. The following5′ oligonucleotide primer (SEQ ID NO:7) and 3′ oligonucleotide primer(SEQ ID NO:8) were first constructed:

SEQ ID NO:7 5′-GCGTCGACCTAGTGACGCTCATACAAATC-3′      <SalI> SEQ ID NO:85′-GCGCGGCCGCTCAGGAGGAGGCTTCCTTGACTG-3′     <-NotI->End\1191            \1172

The 5′ primer corresponds to nucleotides 31-51 from the untranslatedregion of human type II IL-1R clone 75 (SEQ ID NO:1) with a 5′ add-on ofa SalI restriction site; this nucleotide sequence is capable ofannealing to the (−) strand complementary to nucleotides 31-51 of humanclone 75. The 3′ primer is complementary to nucleotides 1191-1172 (whichincludes anti-sense nucleotides encoding 3 amino acids of human type IIIL-1R clone 75 (SEQ ID NO:1) and has a 5′ add-on of a NotI restrictionsite and a stop codon.

The following PCR reagents were added to a 1.5 ml Eppendorf microfugetube: 10 μl of 10×PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3 at 25°C, 15 mM MgCl₂, and 1 mg/ml gelatin) (Perkin-Elmer Cetus, Norwalk,Conn.), 10 μl of a 2 mM solution containing each dNTP (2 mM dATP, 2 mMdCTP, 2 mM dGTP and 2 mM dTTP), 2.5 units (0.5 μl of standard 5000units/ml solution) of Taq DNA polymerase (Perkin-Elmer Cetus), 50 ng oftemplate DNA and 5 μl of a 20 μM solution of each of the aboveoligonucleotide primers and 74.5 μl water to a final volume of 100 μl.The final mixture was then overlaid with 100 μl parafin oil. PCR wascarried out using a DNA thermal cycler (Ericomp, San Diego, Calif.) byinitially denaturing the template at 94° for 90 seconds, reannealing at55° for 75 seconds and extending the cDNA at 72° for 150 seconds. PCRwas carried out for an additional 20 cycles of amplification using astep program (denaturation at 94°, 25 sec; annealing at 55°, 45 sec;extension at 72°, 150 sec.), followed by a 5 minute extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenolchloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10 μl aliquot of the extracted DNA wasseparated by electrophoresis on 1% SeaKem™ agarose (FMC BioProducts,Rockland, Me.) and stained with ethidium bromide to confirm that the DNAfragment size was consistent with the predicted product.

20 μl of the PCR-amplified cDNA products were then digested with SalIand NotI restriction enzymes using standard procedures. The SalI/NotIrestriction fragment was then separated on a 1.2% Seaplaque™ low gellingtemperature (LGT) agarose, and the band representing the fragment wasisolated. The fragment was ligated into the pDC406 vector by a standard“in gel” ligation method, and the vector was transfected into CV1-EBNAcells and expressed as described above in Example 1.

Example 3 Isolation of cDNAs Encoding Murine Type II IL-1R

Murine type II IL-1R cDNAs were isolated from a cDNA library made fromthe murine pre-B cell line 70Z/3 (ATCC TIB 158), by cross specieshybridization with a human Type II IL-1R probe. A cDNA library wasconstructed in a λphage vector using λgt10 arms and packaged in vitro(Gigapack®, Stratagene, San Diego) according to the manufacturer'sinstructions. A double-stranded human Type II IL-1R probe was producedby excising an approximately 1.35 kb SalI restriction fragment of thehuman type II IL-1R clone 75 and ³²P-labelling the cDNA using randomprimers (Boehringer-Mannheim). The murine cDNA library was amplifiedonce and a total of 5×10⁵ plaques were screened with the human probe in35% formamide (5×SSC, 42° C.). Several murine type II IL-1R cDNA clones(including clone λ2) were isolated; however, none of the clones appearedto be full-length. Nucleotide sequence information obtained from thepartial clones was used to clone a full-length murine type II IL-1R cDNAas follows.

A full-length cDNA clone encoding murine type II IL-1R was isolated bythe method of Rapid Amplification of cDNA Ends (RACE) described byFrohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988, using RNA fromthe murine pre-B cell line 70Z/3. Briefly, the RACE method uses PCR toamplify copies of a region of cDNA between a known point in the cDNAtranscript (determined from nucleotide sequence obtained as describedabove) and the 3′ end. An adaptor-primer having a sequence containing 17dT base pairs and an adaptor sequence containing three endonucleaserecognition sites (to place convenient restriction sites at the 3′ endof the cDNA) is used to reverse transcribe a population of mRNA andproduce (−) strand cDNA. A primer complementary to a known stretch ofsequence in the 5′ untranslated region of the murine type II IL-1R clone2 cDNA, described above, and oriented in the 3′ direction is annealedwith the (−) strand cDNA and extended to generate a complementary (+)strand cDNA. The resulting double-strand cDNA is amplified by PCR usingprimers that anneal to the natural 5′-end and synthetic 3′-end poly(A)tail. Details of the RACE procedure are as follows.

The following PCR oligonucleotide primers (d(T)₁₇ adaptor-primer, 5′amplification primer and 3′ amplification primer, respectively) werefirst constructed:

SEQ ID NO:9 5′-CTGCAGGCGGCCGCGGATCC(T)₁₇-3′    <PstI> <-NotI-> <BamHI>SEQ ID NO:10 5′-GCGTCGACGGCAAGAAGCAGCAAGGTAC-3′     <SalI>\15               \34 SEQ ID NO:11 5′-CTGCAGGCGGCCGCGGATCC-3′   <PstI> <-NotI-> <BamHI>

Briefly, the d(T)₁₇ adapter-primer (SEQ ID NO:9) contains nucleotidesequence anneals to the poly(A)+ region of a population mRNA transcriptsand is used to generated (−) strand cDNA reverse transcripts from mRNA;it also contains endonuclease restriction sites for PstI, NotI and BamHIto be introduced into the DNA being amplified by PCR. The 5′amplification primer (SEQ ID NO:10) corresponds to nucleotides 15-34from the 5′ untranslated region of murine type II IL-1R clone λ2 with a5′ add-on of a SalI restriction site; this nucleotide sequence annealsto the (−) strand cDNA generated by reverse transcription with thed(T)₁₇ adaptor-primer and is extended to generate (+) strand cDNA. The3′ primer (SEQ ID NO:11) anneals to the (+) strand DNA having the aboveendonuclease restriction sites and is extended to generate adouble-stranded full-length cDNA encoding murine type II IL-1R, whichcan then be amplified by a standard PCR reaction. Details of the PCRprocedure are as follows.

Poly(A)⁺ mRNA was isolated by oligo dT cellulose chromatography fromtotal RNA extracted from 70Z/3 cells using standard methods described byManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Lab., Cold Spring Harbor, N.Y., 1982) and reverse transcribed asfollows. Approximately 1 μg of poly(A)⁺ mRNA in 16.5 μl of water washeated at 68° C. for 3 minutes and then quenched on ice, and added to 2μl of 10×RTC buffer (500 mM Tris-HCl, pH 8.7 at 22° C., 60 mM MgC12, 400mM KCl, 10 mM DTT, each dNTP at 10 mM), 10 units of RNasin (PromegaBiotech), 0.5 μg of d(T)₁₇-adapter primer and 10 units of AMV reversetranscriptase (Life Sciences) in a total volume of 20 μl, and incubatedfor a period of 2 hours at 42° C. to reverse transcribe the mRNA andsynthesize a pool of cDNA. The reaction mixture was diluted to 1 ml withTE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and stored at 4° C.overnight.

Approximately 1 or 5 μl of the cDNA pool was combined with 5 μl of a 20μM solution of the 5′ amplification primer, containing sequencecorresponding to the sequence of nucleotides 15-34 of murine type IIIL-1R clone λ2, 5 μl of a 20 μM solution of the 3′ amplification primer,10 μl of 10×PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.4, 20° C.), 14mM MgCl₂, and 1 mg/ml gelatin), 4 μl of 5 mM each dNTP (containing 5 mMdATP, 5 mM dCTP, 5 mM dGTP and 5 mM dTTP), 2.5 units (0.5 μl of standard5000 units/ml solution) of Taq DNA polymerase (Perkin-Elmer CetusInstruments), diluted to a volume of 100 μl. The final mixture was thenoverlaid with 100 μl parafin oil. PCR was carried out using a DNAthermal cycler (Perkin-Elmer/Cetus) by initially denaturing the templateat 94° for 90 seconds, reannealing at 64° for 75 seconds and extendingthe cDNA at 72° for 150 seconds. PCR was carried out for an additional25 cycles of amplification using the following step program(denaturation at 94° for 25 sec; annealing at 55° for 45 sec; extensionat 72° for 150 sec.), followed by a 7 minute final extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenol-chloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10 μl aliquot of the extracted DNA wasseparated by electrophoresis on 1% SeaKem™ agarose (FMC BioProducts,Rockland, Me.) and stained with ethidium bromide to confirm that the DNAfragment size was consistent with the predicted product. The gel wasthen blotted and probed with a 5′ 610 bp EcoRI fragment of murine typeII IL-1R clone λ2 from above to confirm that the band contained DNAencoding murine type II IL-1R.

The PCR-amplified cDNA products were then concentrated by centrifugationin an Eppendorf microfuge at full speed for 20 min., followed by ethanolprecipitation in 1/10 volume sodium acetate (3 M) and 2.5 volumeethanol. 30 μl of the concentrate was digested with SalI and NotIrestriction enzymes using standard procedures. The SalI/Notl restrictionfragment was then separated on a 1.2% LGT agarose gel, and the bandrepresenting the fragment was isolated. The restriction fragments werethen purified from the agarose using GeneClean™ (Bio-101, La Jolla,Calif.).

The resulting purified restriction fragment was ligated into the pDC406vector, which was then transfected into CV1-EBNA cells and expressed asdescribed above in Example 1.

The Sequence Listing setting forth the nucleotide (SEQ ID No:12) andpredicted amino acid sequences (SEQ ID No:12 and SEQ ID NO:13) andassociated information appears at the end of the specificationimmediately prior to the claims.

Example 4 Construction and Expression of cDNAs Encoding Murine SolubleType II IL-1R

A cDNA encoding soluble murine type II IL-1R (having the sequence ofamino acids-13-345 of SEQ ID NO:12) was constructed by PCR amplification70Z/3 poly(A)⁺ mRNA as a template and the following procedure asdescribed for the full length clone encoding murine type II IL-1R. Thefollowing PCR oligonucleotide primers (d(T)₁₇ adaptor-primer, 5′amplification primer and 3′ amplification primer, respectively) wereconstructed:

SEQ ID NO:9 5′-CTGCAGGCGGCCGCGGATCC(T)₁₇-3′    <PstI> <-NotI-> <BamHI>SEQ ID NO:10 5′-GCGTCGACGGCAAGAAGCAGCAAGGTAC-3′     <SalI>\15               \34 SEQ ID NO:145′-GCGCGGCCGCCTAGGAAGAGACTTCTTTGACTGTGG-3′   <--NotI-->EndSerSerValGluLysValThrThr

The d(T)₁₇ adaptor-primer and 5′ amplification primer are identical withSEQ ID NO:9 and SEQ ID NO:10, described in Example 5. The 3′ end of SEQID NO:12 is complementary to nucleotides 1145-1166 of SEQ ID NO:12 andhas a 5′ add-on of a NotI restriction site and a stop codon.

A pool of cDNA was synthesized from poly(A)⁺ mRNA using the d(T)₁₇adaptor-primer as described in Example 3. To a 1.5 ml Eppendorfmicrofuge tube was added approximately 1 μl of the cDNA pool, 5 μl of a20 μM solution of the 5′ amplification primer, 5 μl of a 20 μM solutionof the 3′ amplification primer, 10 μL of 10×PCR buffer (500 mM KCl, 100mM Tris-HCl (pH 8.4 at 20° C.), 14 mM MgCl₂, and 1 mg/ml gelatin), 4 μlof 5 mM each of dNTP (containing 5 mM dATP, 5 mM dCTP, 5 mM dGTP and 5ml dTTP), 2.5 units (0.5 μl of standard 5000 units/ml solution) of TaqDNA polymerase (Perkin-Elmer Cetus Instruments), diluted with 75.4 μlwater to a volume of 100 μl. The final mixture was then overlaid with100 μl parafin oil. PCR was carried out using a DNA thermal cycler(Ericomp) by initially denaturing the template at 94° for 90 seconds,reannealing at 55° for 75 seconds and extending the cDNA at 72° for 150seconds. PCR was carried out for an additional 20 cycles ofamplification using the following step program (denaturation at 94° for25 sec; annealing at 55° for 45 sec; extension at 72° for 150 sec.),followed by a 7 minute final extension at 72°.

The sample was removed from the parafin oil and DNA extracted byphenol-chloroform extraction and spun column chromatography over G-50(Boehringer Mannheim). A 10 μl aliquot of the extracted DNA wasseparated by electrophoresis on 1% SeaKem™ agarose (FMC BioProducts,Rockland, Me.) and stained with ethidium bromide to confirm that the DNAfragment size was consistent with the predicted product.

The PCR-amplified cDNA products were then concentrated by centrifugationin an Eppendorf microfuge at full speed for 20 min., followed by ethanolprecipitation in 1/10 volume sodium acetate (3 M) and 2.5 volumeethanol. 50 μl was digested with Sall and NotI restriction enzymes usingstandard procedures. The SalI/NotI restriction fragment was thenseparated on a 1.2% Seaplaque LGT agarose gel, and the band representingthe fragment was isolated. The restriction fragment was then purifiedfrom the isolated band using the following freeze/thaw method. The bandfrom the gel was split into two 175 μl fragments and placed into two 1.5ml Eppendorf microfuge tubes. 500 μl of isolation buffer (0.15 M NaCl,10 mM Tris, pH 8.0, 1 mM EDTA) was added to each tube and the tubesheated to 68° C. to melt the gel. The gels were then frozen on dry icefor 10 minutes, thawed at room temperature and centrifuged at 4° C. for30 minutes. Supernatants were then removed and placed in a new tube,suspended in 2 mL ethanol, and centrifuged at 4° C. for an additional 30minutes to form a DNA pellet. The DNA pellet was washed with 70%ethanol, centrifuged for 5 minutes, removed from the tube andresuspended in 20 μl TE buffer.

The resulting purified restriction fragments were then ligated into thepDC406 vector. A sample of the ligation was transformed into DH5α andcolonies were analyzed to check for correct plasmids. The vector wasthen transfected into COS-7 cells and expressed as described above inExample 1.

Example 5 Type II IL-1R Binding Studies

The binding inhibition constant of recombinant human type II IL-1R,expressed and purified as described in Example 1 above, was determinedby inhibition binding assays in which varying concentrations of acompetitor (IL-1β or IL-1α) was incubated with a constant amount ofradiolabeled IL-1β or IL-1α and cells expressing the type II IL-1R. Thecompetitor binds to the receptor and prevents the radiolabeled ligandfrom binding to the receptor. Binding assays were performed by aphthalate oil separation method essentially as describe by Dower et al.,J. Immunol. 132:751, 1984 and Park et al., J. Biol. Chem. 261:4177,1986. Briefly, CV1/EBNA cells were incubated in six-well plates (Costar,Cambridge, Me.) at 4° C. for 2 hours with ¹²⁵I-IL1β in 1 ml bindingmedium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2%BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2). Sodium azidewas included to inhibit internalization and degradation of ¹²⁵IL-1 bycells at 37° C. The plates were incubated on a gyratory shaker for 1hour at 37° C. Replicate aliquots of the incubation mixture were thentransferred to polyethylene centrifuge tubes containing a phthalate oilmixture comprising 1.5 parts dibutylphthalate, to 1 partbis(s-ethylhexyl)phthalate. Control tubes containing a 100× molar excessof unlabeled IL-1β were also included to determine non-specific binding.The cells with bound ¹²⁵I-IL-1 were separated from unbound 125I-IL-1 bycentrifugation for 5 minutes at 15,000×g in an Eppendorf Microfuge. Theradioactivity associated with the cells was then determined on a gammacounter. This assay (using unlabeled human IL-1β as a competitor toinhibit binding of ¹²⁵I-IL-1β to type II IL-1R) indicated that the fulllength human type II IL-1R exhibits biphasic binding to IL-1β with aK_(I1) of approximately 19±8×10⁹ and K_(I2) of approximately0.2±0.002×10⁹. Using unlabeled human IL-1β to inhibit binding of¹²⁵I-IL-1α to type II IL-1R, the full length human type II IL-1Rexhibited biphasic binding to IL-1β with a K_(I1) of approximately2.0±1×10⁹ and K_(I2) of approximately 0.013±0.003×10⁹.

The binding inhibition constant of the soluble human type II IL-1R,expressed and purified as described in Example 2 above, is determined bya inhibition binding assay in which varying concentrations of an IL-1βcompetitor is incubated with a constant amount of radiolabeled I-IL-1βand CB23 cells (an Epstein Barr virus transformed cord blood Blymphocyte cell line) expressing the type II IL-1R. Binding assays werealso performed by a phtahlate oil separation method essentially asdescribe by Dower et al., J. Immunol. 132:751, 1984 and Park et al., J.Biol. Chem. 261:4177, 1986. Briefly, COS-7 cells were transfected withthe expression vector pDC406 containing a cDNA encoding the solublehuman type II IL-1R described above. Supernatants from the COS cellswere harvested 3 days after transfection and serially diluted in bindingmedium (Roswell Park Memorial Institute (RPMI) 1640 medium containing 2%BSA, 20 mM Hepes buffer, and 0.2% sodium azide, pH 7.2) in 6 well platesto a volume of 50 μl/well. The supernatants were incubated with 50 μl of9×10⁻¹⁰ M ¹²⁵I-IL-1β plus 2.5×10⁶ CB23 cells at 8° C. for 2 hours withagitation. Duplicate 60 μl aliquots of the incubation mixture were thentransferred to polyethylene centrifuge tubes containing a phthalate oilmixture comprising 1.5 parts dibutylphthalate, to 1 partbis(s-ethylhexyl)phthalate. A negative control tube containing 3×10⁻⁶ Munlabeled IL-1β was also included to determine non-specific binding(100% inhibition) and a positive control tube containing 50 ml bindingmedium with only radiolabled IL-1β was included to determine maxiumbinding. The cells with bound ¹²⁵-IL-1β were separated from unbound¹²⁵I-IL-1β by centrifugation for 5 minutes at 15,000×g in an EppendorfMicrofuge. Supernatants containing unbound ¹²⁵I-IL-1β were discarded andthe cells were carefully rinsed with ice-cold binding medium. The cellswere then incubated in 1 ml of trypsin-EDTA at 37° C. for 15 minutes andcells were harvested. The radioactivity of the cells was then determinedon a gamma counter. This inhibition binding assay (using soluble humantype II IL-1R to inhibit binding of IL-1β indicated that the solublehuman type II IL-1R has a K_(I) of approximately 3.5×10⁹ M⁻¹. Inhibitionof IL-1α binding by soluble human type II IL-1R using the same procedureindicated that soluble human type II IL-1R has a K_(I) of 1.4×10⁸ M⁻¹.

Murine type II IL-1R exhibits biphasic binding to IL-1β with a K_(I1 of)0.8×10⁹ and a K_(I2) of less then 0.01×10⁹.

Example 6 Type II IL-1R Affinity Crosslinking Studies

Affinity crosslinking studies were performed essentially as described byPark et al., Proc. Natl. Acad. Sci. USA 84:1669, 1987. Recombinant humanIL-1α and IL-1β used in the assays were expressed, purified and labeledas described previously (Dower et al., J. Exp. Med. 162:501, 1985; Doweret al., Nature 324:266, 1986). Recombinant human IL-1 receptorantagonist (IL-1ra) was cloned using the cDNA sequence published byEisenberg et al., Nature 343:341, 1990, expressed by transienttransfection in COS cells, and purified by affinity chromatography on acolumn of soluble human type I IL-1R coupled to affigel, as described byDower et al., J. Immunol. 143:4314, 1989, and eluted at low pH.

Briefly, CV1/EBNA cells (4×10⁷/ml) expressing recombinant type II IL-1Rwere incubated with ¹²⁵I-IL-1α or ¹²⁵I-IL-1β (1 nM) at 4° C. in thepresence and absence of 1 μM excess of unlabeled IL-1 as a specificitycontrol for 2 hours. The cells were then washed andbis(sulfosuccinimidyl)suberate was added to a final concentration of 0.1mg/ml. After 30 min. at 25° C., the cells were washed and resuspended in100 μl of phosphate-buffered saline (PBS)/1% Triton containing 2 mMleupeptin, 2 mM o-phenanthroline, and 2 mM EGTA to prevent proteolysis.Aliquots of the extract supernatants containing equal amounts (CPM) of¹²⁵I-IL-1 and equal volumes of the specificity controls, were analyzedby SDS/PAGE on a 10% gel using standard techniques.

FIG. 4 shows the results of affinity crosslinking studies conducted asdescribed above, using radiolabeled IL-1α and IL-1β, to compare thesizes of the recombinant murine and human type II IL-1 receptor proteinsto their natural counterparts, and to natural and recombinant murine andhuman type I IL-1 receptors. In general, the sizes of thetransiently-expressed recombinant receptors are similar to the naturalreceptors, although the recombinant proteins migrate slightly faster andas slightly broader bands, possibly as a result of differences inglycosylation patter when over-expressed in CV1/EBNA cells. The resultsalso indicate that the type II IL-1 receptors are smaller than the typeI IL-1 receptors. One particular combination (natural human type Ireceptor with IL-1β) failed to yield specific crosslinking products.Since approximately equal amounts of label were loaded into eachexperimental lane, as indicated by the intensity of the free ligandbands at the bottom of the gels, this combinantion must crosslinkrelatively poorly.

The lane showing natural human type II IL-1 receptor-bearing cellscross-linked with ¹²⁵I-IL-1α reveals a component in the size range(M_(r)=100,000) of complexes with natural and recombinant type Ireceptors. No such complex can be detected in the lane containingrecombinant type II IL-1 receptor, possibly as a result of low levelexpression of type I IL-1 receptors on the CB23 cells, since these cellscontain trace amounts of type I IL-1 receptor mRNA.

Example 7 Preparation of Monoclonal Antibodies to Type II IL-1R

Preparations of purified recombinant type II IL-1R, for example, humantype II IL-1R, or transfected COS cells expressing high levels of typeII IL-1R are employed to generate monoclonal antibodies against type IIIL-1R using conventional techniques, for example, those disclosed inU.S. Pat. No. 4,411,993. Such antibodies are likely to be useful ininterfering with IL-1 binding to type II IL-1R, for example, inameliorating toxic or other undesired effects of IL-1, or as componentsof diagnostic or research assays for IL-1 or soluble type II IL-1R.

To immunize mice, type II IL-1R immunogen is emulsified in completeFreund's adjuvant and injected in amounts ranging from 10-100 μgsubcutaneously and interaperitoneally into Balb/c mice. Ten to twelvedays later, the immunized animals are boosted with additional immunogenemulsified in incomplete Freund's adjuvant and periodically boostedthereafter on a weekly to biweekly immunization schedule. Serum samplesare periodically taken by retro-orbital bleeding or tail-tip excisionfor testing by dot-blot assay (antibody sandwich) or ELISA(enzyme-linked immunosorbent assay), or receptor binding inhibition.Other assay procedures are also suitable. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to the murine myelomacell line NS1 or Ag8.653. Hybridoma cell lines generated by thisprocedure are plated in multiple microtiter plates in a HAT selectivemedium (hypoxanthine, aminopterin, and thymidine) to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith type II IL-1R, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat.No. 4,703,004. Positive clones are then injected into the peritonealcavities of syngeneic Balb/c mice to produce ascites containing highconcentrations (>1 mg/ml) of anti-type II IL-1R monoclonal antibody, orgrown in flasks or roller bottles. The resulting monoclonal antibody canbe purified by ammonium sulfate precipitation followed by gel exclusionchromatography, and/or affinity chromatography based on binding ofantibody to Protein A of Staphylococcus aureus or protein G fromStreptococci.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 and SEQ ID NO:2 show the nucleotide sequence and predictedamino acid sequence of human type II IL-1R. The mature peptide encodedby this sequence is defined by amino acids 1-385. The predicted signalpeptide is defined by amino acids −13 through −1. The predictedtransmembrane region is defined by amino acids 331-356.

SEQ ID NO:3-SEQ ID NO:6 are various oligonucleotides used to clone thefull-length human type II IL-1R.

SEQ ID NO:7 and SEQ ID NO:8 are oligonucleotide primers used toconstruct a soluble human type II IL-1R by polymerase chain reaction(PCR).

SEQ ID NO:9-SEQ ID NO:11 are oligonucleotide primers used to clone afull-length and soluble murine type II IL-1Rs.

SEQ ID NO:12 and SEQ ID NO:13 show the nucleotide sequence and predictedamino acid sequence of the full-length murine type II IL-1R. The maturepeptide encoded by this sequence is defined by amino acids 1-397. Thepredicted signal peptide is defined by amino acids -13 through -1. Thepredicted transmembrane region is defined by amino acids 343-368.

SEQ ID NO:14 is an oligonucleotide primer used to construct a solublemurine type II IL-1R.

14 1357 base pairs nucleic acid double linear cDNA to mRNA N N Homosapiens Human B cell lymphoblastoid CB23 CB23 cDNA pHuIL-1RII75 CDS154..1350 mat_peptide 193..1347 sig_peptide 154..192 1 CTGGAAAATACATTCTGCTA CTCTTAAAAA CTAGTGACGC TCATACAAAT CAACAGAAAG 60 AGCTTCTGAAGGAAGACTTT AAAGCTGCTT CTGCCACGTG CTGCTGGGTC TCAGTCCTCC 120 ACTTCCCGTGTCCTCTGGAA GTTGTCAGGA GCA ATG TTG CGC TTG TAC GTG TTG 174 Met Leu ArgLeu Tyr Val Leu -13 -10 GTA ATG GGA GTT TCT GCC TTC ACC CTT CAG CCT GCGGCA CAC ACA GGG 222 Val Met Gly Val Ser Ala Phe Thr Leu Gln Pro Ala AlaHis Thr Gly -5 1 5 10 GCT GCC AGA AGC TGC CGG TTT CGT GGG AGG CAT TACAAG CGG GAG TTC 270 Ala Ala Arg Ser Cys Arg Phe Arg Gly Arg His Tyr LysArg Glu Phe 15 20 25 AGG CTG GAA GGG GAG CCT GTA GCC CTG AGG TGC CCC CAGGTG CCC TAC 318 Arg Leu Glu Gly Glu Pro Val Ala Leu Arg Cys Pro Gln ValPro Tyr 30 35 40 TGG TTG TGG GCC TCT GTC AGC CCC CGC ATC AAC CTG ACA TGGCAT AAA 366 Trp Leu Trp Ala Ser Val Ser Pro Arg Ile Asn Leu Thr Trp HisLys 45 50 55 AAT GAC TCT GCT AGG ACG GTC CCA GGA GAA GAA GAG ACA CGG ATGTGG 414 Asn Asp Ser Ala Arg Thr Val Pro Gly Glu Glu Glu Thr Arg Met Trp60 65 70 GCC CAG GAC GGT GCT CTG TGG CTT CTG CCA GCC TTG CAG GAG GAC TCT462 Ala Gln Asp Gly Ala Leu Trp Leu Leu Pro Ala Leu Gln Glu Asp Ser 7580 85 90 GGC ACC TAC GTC TGC ACT ACT AGA AAT GCT TCT TAC TGT GAC AAA ATG510 Gly Thr Tyr Val Cys Thr Thr Arg Asn Ala Ser Tyr Cys Asp Lys Met 95100 105 TCC ATT GAG CTC AGA GTT TTT GAG AAT ACA GAT GCT TTC CTG CCG TTC558 Ser Ile Glu Leu Arg Val Phe Glu Asn Thr Asp Ala Phe Leu Pro Phe 110115 120 ATC TCA TAC CCG CAA ATT TTA ACC TTG TCA ACC TCT GGG GTA TTA GTA606 Ile Ser Tyr Pro Gln Ile Leu Thr Leu Ser Thr Ser Gly Val Leu Val 125130 135 TGC CCT GAC CTG AGT GAA TTC ACC CGT GAC AAA ACT GAC GTG AAG ATT654 Cys Pro Asp Leu Ser Glu Phe Thr Arg Asp Lys Thr Asp Val Lys Ile 140145 150 CAA TGG TAC AAG GAT TCT CTT CTT TTG GAT AAA GAC AAT GAG AAA TTT702 Gln Trp Tyr Lys Asp Ser Leu Leu Leu Asp Lys Asp Asn Glu Lys Phe 155160 165 170 CTA AGT GTG AGG GGG ACC ACT CAC TTA CTC GTA CAC GAT GTG GCCCTG 750 Leu Ser Val Arg Gly Thr Thr His Leu Leu Val His Asp Val Ala Leu175 180 185 GAA GAT GCT GGC TAT TAC CGC TGT GTC CTG ACA TTT GCC CAT GAAGGC 798 Glu Asp Ala Gly Tyr Tyr Arg Cys Val Leu Thr Phe Ala His Glu Gly190 195 200 CAG CAA TAC AAC ATC ACT AGG AGT ATT GAG CTA CGC ATC AAG AAAAAA 846 Gln Gln Tyr Asn Ile Thr Arg Ser Ile Glu Leu Arg Ile Lys Lys Lys205 210 215 AAA GAA GAG ACC ATT CCT GTG ATC ATT TCC CCC CTC AAG ACC ATATCA 894 Lys Glu Glu Thr Ile Pro Val Ile Ile Ser Pro Leu Lys Thr Ile Ser220 225 230 GCT TCT CTG GGG TCA AGA CTG ACA ATC CCG TGT AAG GTG TTT CTGGGA 942 Ala Ser Leu Gly Ser Arg Leu Thr Ile Pro Cys Lys Val Phe Leu Gly235 240 245 250 ACC GGC ACA CCC TTA ACC ACC ATG CTG TGG TGG ACG GCC AATGAC ACC 990 Thr Gly Thr Pro Leu Thr Thr Met Leu Trp Trp Thr Ala Asn AspThr 255 260 265 CAC ATA GAG AGC GCC TAC CCG GGA GGC CGC GTG ACC GAG GGGCCA CGC 1038 His Ile Glu Ser Ala Tyr Pro Gly Gly Arg Val Thr Glu Gly ProArg 270 275 280 CAG GAA TAT TCA GAA AAT AAT GAG AAC TAC ATT GAA GTG CCATTG ATT 1086 Gln Glu Tyr Ser Glu Asn Asn Glu Asn Tyr Ile Glu Val Pro LeuIle 285 290 295 TTT GAT CCT GTC ACA AGA GAG GAT TTG CAC ATG GAT TTT AAATGT GTT 1134 Phe Asp Pro Val Thr Arg Glu Asp Leu His Met Asp Phe Lys CysVal 300 305 310 GTC CAT AAT ACC CTG AGT TTT CAG ACA CTA CGC ACC ACA GTCAAG GAA 1182 Val His Asn Thr Leu Ser Phe Gln Thr Leu Arg Thr Thr Val LysGlu 315 320 325 330 GCC TCC TCC ACG TTC TCC TGG GGC ATT GTG CTG GCC CCACTT TCA CTG 1230 Ala Ser Ser Thr Phe Ser Trp Gly Ile Val Leu Ala Pro LeuSer Leu 335 340 345 GCC TTC TTG GTT TTG GGG GGA ATA TGG ATG CAC AGA CGGTGC AAA CAC 1278 Ala Phe Leu Val Leu Gly Gly Ile Trp Met His Arg Arg CysLys His 350 355 360 AGA ACT GGA AAA GCA GAT GGT CTG ACT GTG CTA TGG CCTCAT CAT CAA 1326 Arg Thr Gly Lys Ala Asp Gly Leu Thr Val Leu Trp Pro HisHis Gln 365 370 375 GAC TTT CAA TCC TAT CCC AAG TGA AATAAAT 1357 Asp PheGln Ser Tyr Pro Lys . 380 385 398 amino acids amino acid linear protein2 Met Leu Arg Leu Tyr Val Leu Val Met Gly Val Ser Ala Phe Thr Leu -13-10 -5 1 Gln Pro Ala Ala His Thr Gly Ala Ala Arg Ser Cys Arg Phe Arg Gly5 10 15 Arg His Tyr Lys Arg Glu Phe Arg Leu Glu Gly Glu Pro Val Ala Leu20 25 30 35 Arg Cys Pro Gln Val Pro Tyr Trp Leu Trp Ala Ser Val Ser ProArg 40 45 50 Ile Asn Leu Thr Trp His Lys Asn Asp Ser Ala Arg Thr Val ProGly 55 60 65 Glu Glu Glu Thr Arg Met Trp Ala Gln Asp Gly Ala Leu Trp LeuLeu 70 75 80 Pro Ala Leu Gln Glu Asp Ser Gly Thr Tyr Val Cys Thr Thr ArgAsn 85 90 95 Ala Ser Tyr Cys Asp Lys Met Ser Ile Glu Leu Arg Val Phe GluAsn 100 105 110 115 Thr Asp Ala Phe Leu Pro Phe Ile Ser Tyr Pro Gln IleLeu Thr Leu 120 125 130 Ser Thr Ser Gly Val Leu Val Cys Pro Asp Leu SerGlu Phe Thr Arg 135 140 145 Asp Lys Thr Asp Val Lys Ile Gln Trp Tyr LysAsp Ser Leu Leu Leu 150 155 160 Asp Lys Asp Asn Glu Lys Phe Leu Ser ValArg Gly Thr Thr His Leu 165 170 175 Leu Val His Asp Val Ala Leu Glu AspAla Gly Tyr Tyr Arg Cys Val 180 185 190 195 Leu Thr Phe Ala His Glu GlyGln Gln Tyr Asn Ile Thr Arg Ser Ile 200 205 210 Glu Leu Arg Ile Lys LysLys Lys Glu Glu Thr Ile Pro Val Ile Ile 215 220 225 Ser Pro Leu Lys ThrIle Ser Ala Ser Leu Gly Ser Arg Leu Thr Ile 230 235 240 Pro Cys Lys ValPhe Leu Gly Thr Gly Thr Pro Leu Thr Thr Met Leu 245 250 255 Trp Trp ThrAla Asn Asp Thr His Ile Glu Ser Ala Tyr Pro Gly Gly 260 265 270 275 ArgVal Thr Glu Gly Pro Arg Gln Glu Tyr Ser Glu Asn Asn Glu Asn 280 285 290Tyr Ile Glu Val Pro Leu Ile Phe Asp Pro Val Thr Arg Glu Asp Leu 295 300305 His Met Asp Phe Lys Cys Val Val His Asn Thr Leu Ser Phe Gln Thr 310315 320 Leu Arg Thr Thr Val Lys Glu Ala Ser Ser Thr Phe Ser Trp Gly Ile325 330 335 Val Leu Ala Pro Leu Ser Leu Ala Phe Leu Val Leu Gly Gly IleTrp 340 345 350 355 Met His Arg Arg Cys Lys His Arg Thr Gly Lys Ala AspGly Leu Thr 360 365 370 Val Leu Trp Pro His His Gln Asp Phe Gln Ser TyrPro Lys 375 380 385 24 base pairs nucleic acid single linear DNA(genomic) N N 3 TCGACTGGAA CGAGACGACC TGCT 24 20 base pairs nucleic acidsingle linear DNA (genomic) N Y 4 GACCTTGCTC TGCTGGACGA 20 46 base pairsnucleic acid single linear DNA (genomic) N N 5 CTGTTGGGCT CGCGGTTGAGGACAAACTCT TCGCGGTCTT TCCAGT 46 46 base pairs nucleic acid single linearDNA (genomic) N Y 6 GACAACCCGA GCGCCAACTC CTGTTTGAGA AGCGCCAGAA AGGTCA46 29 base pairs nucleic acid single linear DNA (genomic) N N 7GCGTCGACCT AGTGACGCTC ATACAAATC 29 33 base pairs nucleic acid singlelinear DNA (genomic) N N 8 GCGCGGCCGC TCAGGAGGAG GCTTCCTTGA CTG 33 37base pairs nucleic acid single linear DNA (genomic) N N 9 CTGCAGGCGGCCGCGGATCC TTTTTTTTTT TTTTTTT 37 28 base pairs nucleic acid singlelinear DNA (genomic) N N 10 GCGTCGACGG CAAGAAGCAG CAAGGTAC 28 20 basepairs nucleic acid single linear DNA (genomic) N N 11 CTGCAGGCGGCCGCGGATCC 20 1366 base pairs nucleic acid single linear cDNA to mRNA NN Mouse 70Z/3 70Z/3 l2 CDS 85..1317 mat_peptide 124..1314 sig_peptide85..123 12 GTCGACGGCA AGAAGCAGCA AGGTACAAGA ATACACAGCT CCAGGCTCCAAGGGTCCTGT 60 GCGCTCAGGA AGTTGGTGCG GACA ATG TTC ATC TTG CTT GTG TTA GTAACT 111 Met Phe Ile Leu Leu Val Leu Val Thr -13 -10 -5 GGA GTT TCT GCTTTC ACC ACT CCA ACA GTG GTG CAC ACA GGA AAG GTT 159 Gly Val Ser Ala PheThr Thr Pro Thr Val Val His Thr Gly Lys Val 1 5 10 TCT GAA TCC CCC ATTACA TCG GAG AAG CCC ACA GTC CAT GGA GAC AAC 207 Ser Glu Ser Pro Ile ThrSer Glu Lys Pro Thr Val His Gly Asp Asn 15 20 25 TGT CAG TTT CGT GGC AGAGAG TTC AAA TCT GAA TTG AGG CTG GAA GGT 255 Cys Gln Phe Arg Gly Arg GluPhe Lys Ser Glu Leu Arg Leu Glu Gly 30 35 40 GAA CCT GTG GTT CTG AGG TGCCCC TTG GCA CCT CAC TCC GAC ATC TCC 303 Glu Pro Val Val Leu Arg Cys ProLeu Ala Pro His Ser Asp Ile Ser 45 50 55 60 AGC AGT TCC CAT AGT TTT CTGACC TGG AGT AAA TTG GAC TCT TCT CAG 351 Ser Ser Ser His Ser Phe Leu ThrTrp Ser Lys Leu Asp Ser Ser Gln 65 70 75 CTG ATC CCA AGA GAT GAG CCA AGGATG TGG GTG AAG GGT AAC ATA CTC 399 Leu Ile Pro Arg Asp Glu Pro Arg MetTrp Val Lys Gly Asn Ile Leu 80 85 90 TGG ATT CTG CCA GCA GTG CAG CAA GACTCT GGT ACC TAC ATT TGC ACA 447 Trp Ile Leu Pro Ala Val Gln Gln Asp SerGly Thr Tyr Ile Cys Thr 95 100 105 TTC AGA AAC GCA TCC CAC TGT GAG CAAATG TCT GTG GAA CTC AAG GTC 495 Phe Arg Asn Ala Ser His Cys Glu Gln MetSer Val Glu Leu Lys Val 110 115 120 TTT AAG AAT ACT GAA GCA TCT CTG CCTCAT GTC TCC TAC TTG CAA ATC 543 Phe Lys Asn Thr Glu Ala Ser Leu Pro HisVal Ser Tyr Leu Gln Ile 125 130 135 140 TCA GCT CTC TCC ACC ACC GGG TTACTA GTG TGC CCT GAC CTG AAA GAA 591 Ser Ala Leu Ser Thr Thr Gly Leu LeuVal Cys Pro Asp Leu Lys Glu 145 150 155 TTC ATC TCC AGC AAC GCT GAT GGAAAG ATA CAG TGG TAT AAG GGC GCC 639 Phe Ile Ser Ser Asn Ala Asp Gly LysIle Gln Trp Tyr Lys Gly Ala 160 165 170 ATA CTC TTG GAT AAA GGC AAT AAGGAA TTT CTG AGT GCA GGA GAC CCC 687 Ile Leu Leu Asp Lys Gly Asn Lys GluPhe Leu Ser Ala Gly Asp Pro 175 180 185 ACA CGC CTA TTG ATA TCC AAC ACGTCC ATG GAC GAT GCA GGC TAT TAC 735 Thr Arg Leu Leu Ile Ser Asn Thr SerMet Asp Asp Ala Gly Tyr Tyr 190 195 200 AGA TGT GTT ATG ACA TTT ACC TACAAT GGC CAG GAA TAC AAC ATC ACT 783 Arg Cys Val Met Thr Phe Thr Tyr AsnGly Gln Glu Tyr Asn Ile Thr 205 210 215 220 AGG AAT ATT GAA CTC CGG GTCAAA GGA GCA ACC ACG GAA CCC ATC CCT 831 Arg Asn Ile Glu Leu Arg Val LysGly Ala Thr Thr Glu Pro Ile Pro 225 230 235 GTG ATC ATT TCT CCC CTG GAGACA ATA CCA GCA TCA TTG GGG TCA AGA 879 Val Ile Ile Ser Pro Leu Glu ThrIle Pro Ala Ser Leu Gly Ser Arg 240 245 250 CTG ATA GTC CCG TGC AAA GTGTTT CTG GGA ACT GGT ACA TCT TCC AAC 927 Leu Ile Val Pro Cys Lys Val PheLeu Gly Thr Gly Thr Ser Ser Asn 255 260 265 ACC ATT GTG TGG TGG TTG GCTAAC AGC ACG TTT ATC TCG GCT GCT TAC 975 Thr Ile Val Trp Trp Leu Ala AsnSer Thr Phe Ile Ser Ala Ala Tyr 270 275 280 CCA AGA GGC CGT GTG ACC GAGGGG CTA CAC CAC CAG TAC TCA GAG AAT 1023 Pro Arg Gly Arg Val Thr Glu GlyLeu His His Gln Tyr Ser Glu Asn 285 290 295 300 GAT GAA AAC TAT GTG GAAGTG TCG CTG ATT TTT GAT CCA GTC ACA AGG 1071 Asp Glu Asn Tyr Val Glu ValSer Leu Ile Phe Asp Pro Val Thr Arg 305 310 315 GAG GAT CTG CAT ACA GATTTT AAA TGT GTT GCC TCG AAT CCA CGG AGT 1119 Glu Asp Leu His Thr Asp PheLys Cys Val Ala Ser Asn Pro Arg Ser 320 325 330 TCT CAG TCA CTC CAT ACCACA GTC AAA GAA GTC TCT TCC ACG TTC TCC 1167 Ser Gln Ser Leu His Thr ThrVal Lys Glu Val Ser Ser Thr Phe Ser 335 340 345 TGG AGC ATT GCG CTG GCACCT CTG TCT CTG ATC ATC TTG GTT GTG GGG 1215 Trp Ser Ile Ala Leu Ala ProLeu Ser Leu Ile Ile Leu Val Val Gly 350 355 360 GCA ATA TGG ATG CGC AGACGG TGT AAA CGC AGG GCT GGA AAG ACA TAT 1263 Ala Ile Trp Met Arg Arg ArgCys Lys Arg Arg Ala Gly Lys Thr Tyr 365 370 375 380 GGA CTG ACC AAG CTACGG ACT GAC AAC CAG GAC TTC CCT TCC AGC CCA 1311 Gly Leu Thr Lys Leu ArgThr Asp Asn Gln Asp Phe Pro Ser Ser Pro 385 390 395 AAC TAA ATAAAGGAAATGAAATAAAA AAAAAAAAAA AAAAAGGATC CGCGGCCGC 1366 Asn . 410 amino acidsamino acid linear protein 13 Met Phe Ile Leu Leu Val Leu Val Thr Gly ValSer Ala Phe Thr Thr -13 -10 -5 1 Pro Thr Val Val His Thr Gly Lys Val SerGlu Ser Pro Ile Thr Ser 5 10 15 Glu Lys Pro Thr Val His Gly Asp Asn CysGln Phe Arg Gly Arg Glu 20 25 30 35 Phe Lys Ser Glu Leu Arg Leu Glu GlyGlu Pro Val Val Leu Arg Cys 40 45 50 Pro Leu Ala Pro His Ser Asp Ile SerSer Ser Ser His Ser Phe Leu 55 60 65 Thr Trp Ser Lys Leu Asp Ser Ser GlnLeu Ile Pro Arg Asp Glu Pro 70 75 80 Arg Met Trp Val Lys Gly Asn Ile LeuTrp Ile Leu Pro Ala Val Gln 85 90 95 Gln Asp Ser Gly Thr Tyr Ile Cys ThrPhe Arg Asn Ala Ser His Cys 100 105 110 115 Glu Gln Met Ser Val Glu LeuLys Val Phe Lys Asn Thr Glu Ala Ser 120 125 130 Leu Pro His Val Ser TyrLeu Gln Ile Ser Ala Leu Ser Thr Thr Gly 135 140 145 Leu Leu Val Cys ProAsp Leu Lys Glu Phe Ile Ser Ser Asn Ala Asp 150 155 160 Gly Lys Ile GlnTrp Tyr Lys Gly Ala Ile Leu Leu Asp Lys Gly Asn 165 170 175 Lys Glu PheLeu Ser Ala Gly Asp Pro Thr Arg Leu Leu Ile Ser Asn 180 185 190 195 ThrSer Met Asp Asp Ala Gly Tyr Tyr Arg Cys Val Met Thr Phe Thr 200 205 210Tyr Asn Gly Gln Glu Tyr Asn Ile Thr Arg Asn Ile Glu Leu Arg Val 215 220225 Lys Gly Ala Thr Thr Glu Pro Ile Pro Val Ile Ile Ser Pro Leu Glu 230235 240 Thr Ile Pro Ala Ser Leu Gly Ser Arg Leu Ile Val Pro Cys Lys Val245 250 255 Phe Leu Gly Thr Gly Thr Ser Ser Asn Thr Ile Val Trp Trp LeuAla 260 265 270 275 Asn Ser Thr Phe Ile Ser Ala Ala Tyr Pro Arg Gly ArgVal Thr Glu 280 285 290 Gly Leu His His Gln Tyr Ser Glu Asn Asp Glu AsnTyr Val Glu Val 295 300 305 Ser Leu Ile Phe Asp Pro Val Thr Arg Glu AspLeu His Thr Asp Phe 310 315 320 Lys Cys Val Ala Ser Asn Pro Arg Ser SerGln Ser Leu His Thr Thr 325 330 335 Val Lys Glu Val Ser Ser Thr Phe SerTrp Ser Ile Ala Leu Ala Pro 340 345 350 355 Leu Ser Leu Ile Ile Leu ValVal Gly Ala Ile Trp Met Arg Arg Arg 360 365 370 Cys Lys Arg Arg Ala GlyLys Thr Tyr Gly Leu Thr Lys Leu Arg Thr 375 380 385 Asp Asn Gln Asp PhePro Ser Ser Pro Asn 390 395 36 base pairs nucleic acid single linear DNA(genomic) N N 14 GCGCGGCCGC CTAGGAAGAG ACTTCTTTGA CTGTGG 36

We claim:
 1. An isolated nucleic acid comprising a polynucleotideencoding a polypeptide comprising amino acids 1 through y of SEQ IDNO:2, wherein y is 330 through 385, inclusive.
 2. An isolated nucleicacid comprising a polynucleotide encoding a polypeptide comprising aminoacids 1 through 385 of SEQ ID NO:2.
 3. An isolated nucleic acidcomprising a polynucleotide encoding a polypeptide comprising aminoacids 1 through 333 of SEQ ID NO:2.
 4. An isolated nucleic acidcomprising a polynucleotide encoding a polypeptide comprising aminoacids 1 through 330 of SEQ ID NO:2.
 5. An isolated DNA comprisingnucleotides 193-1191 of SEQ ID NO:1.
 6. A vector comprising apolynucleotide according to claim
 1. 7. A vector comprising apolynucleotide according to claim
 3. 8. A process for preparing apolypeptide, the process comprising culturing a host cell comprising thevector of claim 5 under conditions promoting expression of thepolypeptide.
 9. A process for preparing a polypeptide, the processcomprising culturing a host cell comprising the vector of claim 6 underconditions promoting expression of the polypeptide.
 10. An isolated DNAcomprising nucleotides 154-1191 of SEQ ID NO:1.
 11. An isolated DNAcomprising nucleotides 154-1347 of SEQ ID NO:1.
 12. A host celltransformed with a vector of claim
 7. 13. A process for preparing apolypeptide, the process comprising culturing a host cell comprising theDNA of claim 1 under conditions promoting expression of the polypeptide.14. A host cell transformed with a vector of claim
 6. 15. A process forpreparing a polypeptide, the process comprising culturing a host cellcomprising the DNA of claim 3 under conditions promoting expression ofthe polypeptide.